Divalent nucleobase compounds and uses therefor

ABSTRACT

Described herein are novel divalent nucleobases that each bind two nucleic acid strands, matched or mismatched when incorporated into a nucleic acid or nucleic acid analog backbone (a genetic recognition reagent, or genetic recognition reagent). In one embodiment, the genetic recognition reagent is a peptide nucleic acid (PNA) or gamma PNA (?PNA) oligomer. Uses of the divalent nucleobases and monomers and genetic recognition reagents containing the divalent nucleobases also are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase of International Application No.PCT/US2014/033793 filed Apr. 11, 2014, and claims the benefit ofcopending U.S. Provisional Application Nos. 61/853,758 filed Apr. 11,2013, and 61/854,138 filed Apr. 18, 2013, each of which is incorporatedherein by reference in its entirety.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under Grant No.CHE-1012467 awarded by the National Science Foundation. The governmenthas certain rights in this invention.

BACKGROUND

1. Field of the Invention

Described herein are nucleobases, polymer monomers comprising thenucleobases and nucleic acids and analogs thereof comprising thenucleobases. Also described herein are methods of use of thenucleobases, polymer monomers comprising the nucleobases and nucleicacids and analogs thereof comprising the nucleobases.

2. Description of the Related Art

For most organisms, genetic information is encoded in double-strandedDNA in the form of Watson-Crick base-pairing—in which adenine (A) pairswith thymine (T) and cytosine (C) with guanine (G). Depending on whichset of this genetic information is decoded through transcription andtranslation, the developmental program and physiological status will bedetermined. Development of molecules that can be tailor-designed to bindsequence-specifically to any part of this genetic biopolymer (DNA orRNA), thereby enabling the control of the flow of genetic informationand assessment and manipulation of the genome's structures andfunctions, is important for biological and biomedical research in theeffort to unravel the molecular basis of life, including molecular toolsfor basic research in biology. This effort is also important formedicinal and therapeutic applications for the treatment and detectionof genetic diseases.

Compared to proteins, RNA molecules are easier to target because theyare made up of just four building blocks (A, C, G, U), whoseinteractions are defined by the well-established rules of Watson-Crickbase-pairing. Compared to standard, double-stranded DNA (or RNA), thesecondary structures of RNA are generally thermodynamically less stableand, thus, energetically less demanding for binding because, in additionto being canonical (perfectly-matched) base-pairs, many of them arenoncanonical (mismatched) and contain single-stranded loops, bulges, andjunctions. The presence of these local interacting domains is essentialfor ‘tertiary’ interactions and assembly of the secondary structuresinto compact three-dimensional shapes. As such, slight variations in theinteraction patterns or bonding strengths within these regions will havea profound effect on the overall three-dimensional folding patterns ofRNA. Thus, molecules that can be used to modulate RNA interactions andthereby interfere with the RNA folding behaviors are important asmolecular tools for assessing RNA functions, as well as therapeutic anddiagnostic reagents.

RNA-RNA and RNA-protein interactions play key roles in gene regulation,including replication, translation, folding and packaging. The abilityto selectively bind to regions within the secondary structures of RNAwill often modify their physiological functions.

SUMMARY

Provided herein are reagents that can be used to target double-strandednucleic acid sequences, including RNA secondary structures andmismatches. The reagents are relatively small in size, can bemanufactured in large quantity and more cheaply using solution-phasemethodology, and are readily taken-up by cells. They are especiallyappealing for targeting rapidly evolving sites, such as those associatedwith the pathology of cancer, bacterial and viral infection, because thedescribed recognition scheme is modular in nature and can be readilymodified to match the newly emerged sequence at will. This is a nichethat is not currently fulfilled by small-molecule drugs, or traditionalantisense or antigene targeting approach.

“Janus” nucleobases (JBs) are described herein. Janus nucleobases arecapable of forming directional hydrogen bonding interactions with bothstrands of the DNA and/or RNA double helix whether or not mismatches arepresent. This platform has applications in basic research in biology andbiotechnology, diagnostics and therapeutics. In one embodiment, thenucleobases are attached to a γPNA backbone, integrating conformationalpreorganization inherent in the backbone of γPNA with improvements inhydrogen-bonding and base-stacking capabilities, a common property ofthe Janus nucleobases described herein, into a single system. Unlike thenatural nucleobases, which can only hybridize to one strand of the DNAor RNA double helix, these ‘Janus’ nucleobases can hybridize to bothstrands of the DNA or RNA targets. This method and platform can affectthe regulation of gene expression and modulation of nucleic acidinteractions—as molecular tools for basic research as well astherapeutic and diagnostic reagents for the treatment and detection ofgenetic diseases and pathogenic infections.

Provided herein is a genetic recognition reagent, such as a nucleicacid, gamma peptide nucleic acid (γPNA) or other nucleic acid. Thegenetic recognition reagent comprises a plurality of nucleobase residuesattached to a nucleic acid or nucleic acid analog backbone. At least onenucleobase is a divalent nucleobase chosen from the nucleobasesdescribed in Table A, below, in which each instance of R1 is,independently, a protecting group or H and X is CH or N. Non-limitingexamples of protecting groups include: methyl, formyl, ethyl, acetyl,anisyl, benzyl, benzoyl, carbamate, trifluoroacetyl, diphenylmethyl,triphenylmethyl, N-hydroxysuccinimide, benzyloxymethyl,benzyloxycarbonyl, 2-nitrobenzoyl, t-Boc (tert-butyloxycarbonyl),4-methylbenzyl, 4-nitrophenyl, 2-chlorobenzyloxycarbonyl,2-bromobenzyloxycarbonyl, 2,4,5-trichlorophenyl, thioanizyl, thiocresyl,cbz (carbobenzyloxy), p-methoxybenzyl carbonyl,9-fluorenylmethyloxycarbonyl, pentafluorophenyl, p-methoxybenzyl,3,4-dimethozybenzyl, p-methoxyphenyl, 4-toluenesulfonyl,p-nitrobenzenesulfonates, 9-fluorenylmethyloxycarbonyl,2-nitrophenylsulfenyl, 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl, andp-bromobenzene sulfonyl. In certain embodiments, the backbone is chosenfrom one of a DNA, RNA, peptide nucleic acid (PNA), phosphorothioate,locked nucleic acid, unlocked nucleic acid, 2′-O-methyl-substituted RNA,morpholino nucleic acid, threose nucleic acid, or glycol nucleic acidbackbone, or any combination thereof. In one embodiment, the backbone isa peptide nucleic acid (PNA) backbone, for example and withoutlimitation a γPNA backbone. The backbone is optionally PEGylated, withone or more PEG moieties of two to fifty (—O—CH₂—CH₂—) residues. Anexemplary γPNA backbone includes a backbone monomer residue that is

where R1, R2 and R3 are, independently, H, amino acid side chains,linear or branched (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₈)hydroxyalkyl, (C₃-C₈)aryl, (C₃-C₈)cycloalkyl,(C₃-C₈)aryl(C₁-C₆)alkylene, (C₃-C₈)cycloalkyl(C₁-C₆)alkylene, PEGylatedmoieties of the preceding comprising from 1 to 50 (—O—CH₂—CH₂—)residues, —CH₂—(OCH₂—CH₂)_(q)OP₁, —CH₂—(OCH₂—CH₂)_(q)—NHP₁,—CH₂—(OCH₂—CH₂-0)_(q)—SP₁ and —CH₂—(SCH₂—CH₂)_(q)—SP₁,—CH₂—(OCH₂—CH₂)_(r)—OH, —CH₂—(OCH₂—CH₂)_(r)—NH₂,—CH₂—(OCH₂—CH₂)_(r)—NHC(NH)NH₂, or—CH₂—(OCH₂—CH₂)_(r)—S—S[CH₂CH₂]_(s)NHC(NH)NH₂, where P₁ is selected fromthe group consisting of H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₃-C₈)aryl, (C₃-C₈)cycloalkyl, (C₃-C₈)aryl(C₁-C₆)alkylene and(C₃-C₈)cycloalkyl(C₁-C₆)alkylene; q is an integer from 0 to 10,inclusive; r and s are each independently integers from 1 to 50,inclusive; where R1 and R2 are different and one of R1 or R2 is H. Inone example R2 is H, R1 is an amino acid side chain that is optionallyPEGylated, with one or more PEG moieties of one to twelve (—O—CH₂—CH₂—)residues. The nucleobases are arranged in certain embodiments in asequence complementary to a target sequence of a nucleic acid. In oneembodiment, the genetic recognition reagent has 3 to 25 nucleobases. Inone embodiment of the composition described above the geneticrecognition reagent has the structure:

where each instance of R4 is a backbone monomer residue and eachinstance of R is a nucleobase, where at least one instance of R thedivalent nucleobase, E are independently end groups, and “n” is zero ora positive integer ranging from 1 to 48, in which the sequence ofnucleobases R is complementary to a target sequence of a nucleic acid.In another embodiment the divalent nucleobases are, independently chosenfrom JB1, JB2, JB3 and JB4. In yet another embodiment, all instances ofR1 are H.

Also provided is a monomer for production of a genetic recognitionreagent comprising a backbone monomer for a genetic recognition reagentcovalently attached to a divalent nucleobase chosen from those listed inTable A, in which each instance of R1 is, independently, a protectinggroup or H and X is CH or N. In one example, the nucleobase is one ofJB1, JB2, JB3 or JB4. In another embodiment, R1 is a protecting group,such as, for example and without limitation, a protecting group ischosen from one or more of methyl, formyl, ethyl, acetyl, anisyl,benzyl, benzoyl, carbamate, trifluoroacetyl, diphenylmethyl,triphenylmethyl, N-hydroxysuccinimide, benzyloxymethyl,benzyloxycarbonyl, 2-nitrobenzoyl, t-Boc (tert-butyloxycarbonyl),4-methylbenzyl, 4-nitrophenyl, 2-chlorobenzyloxycarbonyl,2-bromobenzyloxycarbonyl, 2,4,5-trichlorophenyl, thioanizyl, thiocresyl,cbz (carbobenzyloxy), p-methoxybenzyl carbonyl,9-fluorenylmethyloxycarbonyl, pentafluorophenyl, p-methoxybenzyl,3,4-dimethozybenzyl, p-methoxyphenyl, 4-toluenesulfonyl,p-nitrobenzenesulfonates, 9-fluorenylmethyloxycarbonyl,2-nitrophenylsulfenyl, 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl, andp-bromobenzenesulfonyl. In one embodiment, the monomer is a peptidenucleic acid (PNA) monomer, and in another, γPNA. In yet anotherembodiment, the backbone monomer is a PNA or γPNA that is PEGylated,with one or more PEG moieties of two to fifty (—O—CH₂—CH₂—) residues. Ina further embodiment, the monomer is a γPNA monomer having thestructure:

where R1, R2 and R4 are, independently, H, amino acid side chains,linear or branched (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₈)hydroxyalkyl, (C₃-C₈)aryl, (C₃-C₈)cycloalkyl,(C₃-C₈)aryl(C₁-C₆)alkylene, (C₃-C₈)cycloalkyl(C₁-C₆)alkylene, PEGylatedmoieties of the preceding comprising from 1 to 50 (—O—CH₂—CH₂—)residues, —CH₂—(OCH₂—CH₂)_(q)—OP₁, —CH₂—(OCH₂—CH₂)_(q)—NHP₁,—CH₂—(OCH₂—CH₂-0)_(q)—SP₁ and —CH₂—(SCH₂—CH₂)_(q)—SP₁,—CH₂—(OCH₂—CH₂)_(r)—OH, —CH₂—(OCH₂—CH₂)_(r)—NH₂,—CH₂—(OCH₂—CH₂)_(r)—NHC(NH)NH₂, or—CH₂—(OCH₂—CH₂)_(r)—S—S[CH₂CH₂]_(s)NHC(NH)NH₂, where P₁ is selected fromthe group consisting of H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₃-C₈)aryl, (C₃-C₈)cycloalkyl, (C₃-C₈)aryl(C₁-C₆)alkylene and(C₃-C₈)cycloalkyl(C₁-C₆)alkylene; q is an integer from 0 to 10,inclusive; r and s are each independently integers from 1 to 50,inclusive; where R1 and R2 are different, and one of R1 or R2 is H, andR3 is H or a protecting group. In one non-limiting example, R2 is H, R1is an amino acid side chain that is optionally PEGylated, with one ormore PEG moieties of one to twelve (—O—CH₂—CH₂—) residues, and R3 is aprotecting group.

In another embodiment, a divalent nucleobase is provided, chosen fromthe divalent nucleobases of Table A, in which each instance of R1 is,independently a protecting group or H and R is a reactive group. Inanother embodiment, the nucleobase is chosen from one the following:

in which each instance of R1 is, independently a protecting group or Hand R is carboxyl.

In another embodiment, a kit is provided comprising any monomer asdescribed herein in a vessel. In one embodiment, the kit comprisesmonomers comprising each of JB1, JB2, JB3, JB4, JB5, JB6, JB7, JB8, JB9,JB9b, JB10, JB11, JB12, JB13, JB14, JB15, JB16 nucleobases, with eachdifferent monomer in a separate vessel. An array comprising a geneticrecognition reagent as described herein also is provided.

A method of detection of a target sequence in a nucleic acid sample isprovided. The method comprising contacting a genetic recognition reagentdescribed herein with a sample comprising nucleic acid and detectingbinding of the genetic recognition reagent with a nucleic acid.

Lastly, a method of isolation and purification or a nucleic acidcontaining a target sequence is provided. The method comprising,contacting a nucleic acid sample with a genetic recognition reagent asdescribed herein, separating the nucleic acid sample from the geneticrecognition reagent, leaving any nucleic acid bound to the geneticrecognition reagent bound to the genetic recognition reagent, andseparating the genetic recognition reagent from any nucleic acid boundto the genetic recognition reagent. In one embodiment of the method, thegenetic recognition reagent is immobilized on a substrate. The methodcomprising contacting a nucleic acid with the substrate, washing thesubstrate to remove unbound nucleic acid from the substrate, but leavingbound nucleic acid bound to the substrate, and eluting the bound nucleicacid from the substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates hydrogen-bonding interactions between (A) naturalbase-pairs, (B) JB 1-4 (labeled 1-4) and the perfectly-matched DNA orRNA target and (C) JB 5-16 (labeled 5-16) and the mismatched DNA or RNAtarget.

FIG. 2 provides structures of exemplary nucleobases.

FIGS. 3A-3F provide exemplary structures for nucleic acid analogs.

FIG. 4 provides examples of amino acid side chains.

FIG. 5 illustrates simulated structures γPNAJB1-4 invading DNA doublehelix.

FIG. 6 illustrates simulated structures of γPNAJB1-16 invading an RNAsecondary structure containing perfectly-matched and mismatchedbase-pairs.

FIG. 7 illustrates exemplary schemes for synthesis of the describeddivalent nucleobases.

FIG. 8 illustrates an exemplary scheme for the synthesis of γPNAmonomers with divalent nucleobases.

FIG. 9 illustrates an exemplary scheme for synthesis of γPNA oligomerswith divalent nucleobases.

DETAILED DESCRIPTION

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges are both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, unless indicated otherwise, the disclosure of ranges is intendedas a continuous range including every value between the minimum andmaximum values. As used herein “a” and “an” refer to one or more.

As used herein, the term “comprising” is open-ended and may besynonymous with “including”, “containing”, or “characterized by”. Theterm “consisting essentially of” limits the scope of a claim to thespecified materials or steps and those that do not materially affect thebasic and novel characteristic(s) of the claimed invention. The term“consisting of” excludes any element, step, or ingredient not specifiedin the claim. As used herein, embodiments “comprising” one or morestated elements or steps also include, but are not limited toembodiments “consisting essentially of” and “consisting of” these statedelements or steps.

Provided herein are nucleic acids and analogs thereof, collectively“genetic recognition reagents” (genetic recognition reagent), that bindspecifically to two nucleic acid strands, whether or not the two strandsare independent strands, two portions of a single strand (e.g., in ahairpin) or contain mismatches in the sense that at one or morepositions within the two strands at the site of binding to the geneticrecognition reagents, the bases are not able to base pair according totraditional Watson-Crick base pairing (A-T/U, T/U-A, G-C or C-G). In oneembodiment, the two strands binding the genetic recognition reagent arenon-complementary, meaning they do not hybridize under physiologicalconditions and typically contain less than 50% complementarity, meaningthat less than 50% of the bases in the two strands are mismatched whenaligned to nucleobases of the genetic recognition reagent. Thus,depending upon choice of nucleobases in the sequence, the geneticrecognition reagents described herein can invade or otherwise hybridizeto two strands of fully-complementary, partially-complementary ornon-complementary double-stranded nucleic acids.

In one embodiment, the genetic recognition reagents described hereincomprise all divalent nucleobases. In another embodiment, the geneticrecognition reagents described herein comprise at least one divalentnucleobases, with other nucleobases being monovalent. As used herein, amonovalent nucleobase binds one nucleobase on a single nucleic acidstrand, while a divalent nucleobase, e.g., a Janus base describedherein, binds to two nucleobases, one on a first nucleic acid strand,and another on a second nucleic acid strand.

Thus in one embodiment, divalent nucleobases are provided. Thosenucleobases can be incorporated into a genetic recognition reagentmonomer, which can then be incorporated into an oligomer of monomerswith a desired sequence of nucleobases. Table A provides exemplarydivalent “Janus” bases, their binding specificities (see also, FIG. 1),structures for the nucleobases, and structures for the nucleobasesresidue as it is attached to a nucleotide and/or a genetic recognitionreagent. In the context of the present disclosure, a “nucleotide” refersto a monomer comprising at least nucleobases and a backbone element,which in a nucleic acid, such as RNA or DNA is ribose or deoxyribose.“Nucleotides” also typically comprise reactive groups that permitpolymerization under specific conditions. In native DNA and RNA, thosereactive groups are the 5′ phosphate and 3′ hydroxyl groups. Forchemical synthesis of nucleic acids and analogs thereof, the bases andbackbone monomers may contain modified groups, such as blocked amines,as are known in the art. A “nucleotide residue” refers to a singlenucleotide that is incorporated into an oligonucleotide orpolynucleotide. Likewise, a “nucleobases residue” refers to anucleobases incorporated into a nucleotide or a nucleic acid or analogthereof. A “genetic recognition reagent” refers generically to a nucleicacid or a nucleic acid analog that comprises a sequence of nucleobasesthat is able to hybridize to a complementary nucleic acid sequence on anucleic acid by cooperative base pairing, e.g., Watson-Crick basepairing or Watson-Crick-like base pairing. Of note, JB1-JB4 bindcomplementary bases (C-G, G-C, A-T and T-A), while JB5-JB16 bindmismatches, and thus can be used to bind two strands of matched and/ormismatched bases.

TABLE A Divalent Nucleobases Bases Nucleobase represented NucleobaseNucleobase residue JB1 T/D*

JB2 D/T

JB3 G/C

JB4 C/G

JB5 C/C

JB6 U/U

JB7 G/G

JB8 D/D

JB9 A/C

JB9b A/C

JB10 C/A

JB11 U/G

JB12 G/U

JB13 C/U

JB14 U/C

JB15 G/D

JB16 D/G

*diaminopurine, an adenine analog

In Table A, R is a reactive group that reacts with a backbone monomerduring synthesis of a monomer. Non-limiting examples of a reactive groupis a carboxyl (e.g., —C—C(O)OH), hydroxyl (e.g., —C—OH), cyanate (e.g.,—C—C≡N), thiol (e.g., —C—SH), (CH₂)_(n)CO₂H or (CH₂)_(n)CO₂Y (n=1-5,Y=any leaving group such as Cl, alkyl, aryl, etc.). X is either CH or N.R1s are each, independently: H or a protecting group. In one embodiment,all instances of X are C(═CH—) and all instances are R1. Where allinstances of R1 are H, the compounds or moieties are said to bedeprotected. Depending on the chemistries employed to prepare themonomers or oligomers comprising the monomers, one or more of R1 isoptionally protected using a protecting group, as is needed. Protectinggroups for amines, include, for example and without limitation: methyl,formyl, ethyl, acetyl, anisyl, benzyl, benzoyl, carbamate,trifluoroacetyl, diphenylmethyl, triphenylmethyl, N-hydroxysuccinimide,benzyloxymethyl, benzyloxycarbonyl, 2-nitrobenzoyl, t-Boc(tert-butyloxycarbonyl), 4-methylbenzyl, 4-nitrophenyl,2-chlorobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl,2,4,5-trichlorophenyl, thioanizyl, thiocresyl, cbz (carbobenzyloxy),p-methoxybenzyl carbonyl, 9-fluorenylmethyloxycarbonyl,pentafluorophenyl, p-methoxybenzyl, 3,4-dimethozybenzyl,p-methoxyphenyl, 4-toluenesulfonyl, p-nitrobenzenesulfonates,9-fluorenylmethyloxycarbonyl, 2-nitrophenylsulfenyl,2,2,5,7,8-pentamethyl-chroman-6-sulfonyl, and p-bromobenzenesulfonyl.JB9b is a structural variation of JB9.

The structure,

is generic to JB4 and JB10, wherein R is

The structure,

is generic to JB8, JB15 and JB 16, where:A is

and B is

where either A is

or B is

and X is CH or N.

The nucleobases of Table A have divalent binding affinity, as indicated.FIG. 1 depicts the hydrogen bonding of the sixteen divalent nucleobasesof Table A, while comparing to Watson-Crick-like hydrogen-bondinginteractions with natural bases. Of note, JB1, JB2, JB3 and JB4 accountfor matched (complementary) sequences, while the remainder of thecompounds bind all other iterations of mis-matched sequences.

Nucleobases are recognition moieties that bind specifically to one ormore of adenine, guanine, thymine, cytosine, and uracil, e.g., byWatson-Crick or Watson-Crick-like base pairing by hydrogen bonding. A“nucleobase” includes primary nucleobases: adenine, guanine, thymine,cytosine, and uracil, as well as modified purine and pyrimidine bases,such as, without limitation, hypoxanthine, xanthene, 7-methylguanine, 5,6, dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine. FIG. 2also depicts non-limiting examples of nucleobases, including monovalentnucleobases (e.g., adenine, cytosine, guanine, thymine or uracil, whichbind to one strand of nucleic acid or nucleic acid analogs), and “clamp”nucleobases, such as a “G-clamp,” which binds complementary nucleobaseswith enhanced strength. Additional purine, purine-like, pyrimidine andpyrimidine-like nucleobases are known in the art, for example asdisclosed in U.S. Pat. Nos. 8,053,212, 8,389,703, and 8,653,254.

Also provided herein are nucleotides having the structure A-B wherein Ais a backbone monomer and B is a divalent nucleobase as described above.The backbone monomer can be any suitable nucleic acid backbone monomer,such as a ribose triphosphate or deoxyribose triphosphate, or a monomerof a nucleic acid analog, such as peptide nucleic acid (PNA), such as agamma PNA (γPNA). The backbone monomer includes both the structural“residue” component, such as the ribose in RNA, and any active groupsthat are modified in linking monomers together, such as the 5′triphosphate and 3′ hydroxyl groups of a ribonucleotide, which aremodified when polymerized into RNA to leave a phosphodiester linkage.Likewise for PNA, the C-terminal carboxyl and N-terminal amine activegroups of the N-(2-aminoethyl)glycine backbone monomer are condensedduring polymerization to leave a peptide (amide) bond. In anotherembodiment, the active groups are phosphoramidite groups useful forphosphoramidite oligomer synthesis, as is broadly-known in the arts. Thenucleotide also optionally comprises one or more protecting groups asare known in the art, such as 4,4′-dimethoxytrityl (DMT), and asdescribed herein. A number of additional methods of preparing syntheticgenetic recognition reagents are known, and depend on the backbonestructure and particular chemistry of the base addition process.Determination of which active groups to utilize in joining nucleotidemonomers and which groups to protect in the bases, and the requiredsteps in preparation of oligomers is well within the abilities of thoseof ordinary skill in the chemical arts and in the particular field ofnucleic acid and nucleic acid analog oligomer synthesis.

Non-limiting examples of common nucleic acid analogs include peptidenucleic acids, such as γPNA, phosphorothioate (e.g., FIG. 3A), lockednucleic acid (2′-O-4′-C-methylene bridge, including oxy, thio or aminoversions thereof, e.g., FIG. 3B), unlocked nucleic acid (the C2′-C3′bond is cleaved, e.g., FIG. 3C), 2′-O-methyl-substituted RNA, morpholinonucleic acid (e.g., Fig_D), threose nucleic acid (e.g., FIG. 3E), glycolnucleic acid (e.g., FIG. 3F, showing R and S Forms), etc. FIG. 3A-Fshows monomer structures for various examples of nucleic acid analogs.FIGS. 3A-3F each show two monomer residues incorporated into a longerchain as indicated by the wavy lines. Incorporated monomers are referredto herein as “residues” and the part of the nucleic acid or nucleic acidanalog oligomer or polymer excluding the nucleobases is referred to asthe “backbone” of the nucleic acid or nucleic acid analog. As anexample, for RNA, an exemplary nucleobase is adenine, a correspondingmonomer is adenosine triphosphate, and the incorporated residue is anadenosine monophosphate residue. For RNA, the “backbone” consists ofribose subunits linked by phosphates, and thus the backbone monomer isribose triphosphate prior to incorporation and a ribose monophosphateresidue after incorporation.

According to one embodiment, with the advent ofconformationally-preorganized γPNA, precise sequence selection is nolonger an issue (Bahal, R., et al. “Sequence-unrestricted, Watson-Crickrecognition of double helical B-DNA by (R)-MiniPEG-γPNAs (2012)ChemBioChem 13:56-60). γPNA can be designed to bind to any sequence ofdouble helical B-DNA based on the well-established rules of Watson-Crickbase-pairing. However, with an arsenal of only natural nucleobases asrecognition elements, strand invasion of DNA by γPNA is confined tosub-physiological ionic strengths (Rapireddy, S., R. et al. “Strandinvasion of mixed-sequence, double-helical B-DNA by γ-peptide nucleicacids containing G-clamp nucleobases under physiological conditions,”(2011) Biochemistry 50:3913-3918). A reduction in the efficiency ofproductive γPNA binding at elevated ionic strengths is not due to thelack of base-pair accessibility, but rather due to the lack of bindingfree energy. Under physiological conditions, DNA double helix issufficiently dynamic to permit strand invasion, provided that therequired binding free energy could be met. One way to improve thebinding free energy of such a system would be to enhance thebase-stacking and H-bonding capabilities of the recognition elements,which is met by the present invention. In one embodiment, γPNA monomersand oligomers containing a specialized set of divalent “Janus'”nucleobases (JBs) that are capable of forming directional hydrogenbonding interactions with both strands of the DNA or RNA double helix isperformed. Examples of the chemical structures of the JBs areillustrated in Table A. Non-limiting examples of γPNA monomers andoligomers are provided below, with, e.g., an amino acid side chain, or aPEGylated (polyethyleneglycol, or PEG) group at the chiral gamma carbon.

As used herein, the term “nucleic acid” refers to deoxyribonucleic acids(DNA) and ribonucleic acids (RNA). Nucleic acid analogs include, forexample and without limitation: 2′-O-methyl-substituted RNA, lockednucleic acids, unlocked nucleic acids, triazole-linked DNA, peptidenucleic acids, morpholino oligomers, dideoxynucleotide oligomers, glycolnucleic acids, threose nucleic acids and combinations thereof including,optionally ribonucleotide or deoxyribonucleotide residue(s). Herein,“nucleic acid” and “oligonucleotide”, which is a short, single-strandedstructure made of up nucleotides, are used interchangeably. Anoligonucleotide may be referred to by the length (i.e. number ofnucleotides) of the strand, through the nomenclature “-mer”. Forexample, an oligonucleotide of 22 nucleotides would be referred to as a22-mer.

A “peptide nucleic acid” refers to a DNA or RNA analog or mimic in whichthe sugar phosphodiester backbone of the DNA or RNA is replaced by aN-(2-aminoethyl)glycine unit. A gamma PNA (γPNA) is an oligomer orpolymer of gamma-modified N-(2-aminoethyl)glycine monomers of thefollowing structure:

where at least one of R1 or R2 attached to the gamma carbon is not ahydrogen, or R1 and R2 are different, such that the gamma carbon is achiral center. When R1 and R2 are hydrogen (N-(2-aminoethyl)-glycinebackbone), or the same, there is no such chirality about the gammacarbon. When R1 and R2 are different, such as when one of R1 or R2 are Hand the other is not, there is chirality about the gamma carbon.Typically, for γPNAs and γPNA monomers, either of R1 or R2 is an H andthe other is an amino acid sidechain or an organic group, such as a(C₁-C₁₀) organic group or hydrocarbon, optionally PEGylated with from 1to 50 oxyethylene residues—that is, [—O—CH₂—CH₂—]_(n), where n is 1 to50, inclusive. R4 can be H or an organic group, such as a (C₁-C₁₀)organic group or hydrocarbon, optionally PEGylated with from 1 to 50oxyethylene residues. For example and without limitation, R1, R2 and R4are, independently, H, amino acid side chains, linear or branched(C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)aryl, (C₃-C₈)cycloalkyl, (C₃-C₈)aryl(C₁-C₆)alkylene,(C₃-C₈)cycloalkyl(C₁-C₆)alkylene, PEGylated moieties of the precedingcomprising from 1 to 50 (—O—CH₂—CH₂—) residues, —CH₂—(OCH₂—CH₂)_(q)OP₁,—CH₂—(OCH₂—CH₂)_(q)—NHP₁, —CH₂—(OCH₂—CH₂-0)_(q)—SP₁ and—CH₂—(SCH₂—CH₂)_(q)—SP₁, —CH₂—(OCH₂—CH₂)_(r)—OH,—CH₂—(OCH₂—CH₂)_(r)—NH₂, —CH₂—(OCH₂—CH₂)_(r)—NHC(NH)NH₂, or—CH₂—(OCH₂—CH₂)_(r)—S—S[CH₂CH₂]_(s)NHC(NH)NH₂, where P₁ is selected fromthe group consisting of H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₃-C₈)aryl, (C₃-C₈)cycloalkyl, (C₃-C₈)aryl(C₁-C₆)alkylene and(C₃-C₈)cycloalkyl(C₁-C₆)alkylene; q is an integer from 0 to 10,inclusive; r and s are each independently integers from 1 to 50,inclusive; where R1 and R2 are different, and optionally one of R1 or R2is H. R3 is H or a protective group

A γPNA monomer incorporated into a γPNA oligomer or polymer,

is referred to herein as a “γPNA monomer residue”, with each residuehaving the same or different R group as its nucleobase, such as adenine,guanine, cytosine, thymine and uracil bases, or other nucleobases, suchas the monovalent and divalent bases described herein, such that theorder of bases on the γPNA is its “sequence”, as with DNA or RNA. Thedepicted γPNA monomer and residue structures show a backbone monomer,and a backbone monomer residue, respectively, attached to a nucleobase(R). A sequence of nucleobases in a nucleic acid or a nucleic acidanalog oligomer or polymer, such as a γPNA oligomer or polymer, binds toa complementary sequence of adenine, guanine, cytosine, thymine and/oruracil residues in a nucleic acid strand by cooperative bonding,essentially as with Watson-Crick binding of complementary bases indouble-stranded DNA or RNA. “Watson-Crick-like” bonding refers tohydrogen bonding of nucleobases other than G, A, T, C or U, such as thebonding of the divalent bases shown herein with G, A, T, C, U or othernucleobases.

An “amino acid side chain” is a side chain for an amino acid. Aminoacids have the structure:

where R is the amino acid side chain. Non-limiting examples of aminoacid side chains are shown in FIG. 4. Glycine is not represented becausein the embodiment R1s are both H.

The following are exemplary definitions of various moieties or groups asused herein. “Alkyl” refers to straight, branched chain, or cyclichydrocarbon groups including from 1 to about 20 carbon atoms, forexample and without limitation C₁₋₃, C₁₋₆, C₁₋₁₀ groups, for example andwithout limitation, straight, branched chain alkyl groups such asmethyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl, and the like. “Substituted alkyl” refers toalkyl substituted at 1 or more, e.g., 1, 2, 3, 4, 5, or even 6positions, which substituents are attached at any available atom toproduce a stable compound, with substitution as described herein.“Optionally substituted alkyl” refers to alkyl or substituted alkyl.“Halogen,” “halide,” and “halo” refers to —F, —CI, —Br, and/or —I.“Alkylene” and “substituted alkylene” refer to divalent alkyl anddivalent substituted alkyl, respectively, including, without limitation,ethylene (—CH₂—CH₂—). “Optionally substituted alkylene” refers toalkylene or substituted alkylene.

“Alkene or alkenyl” refers to straight, branched chain, or cyclichydrocarbyl groups including from 2 to about 20 carbon atoms, such as,without limitation C₁₋₃, C₁₋₆, C₁₋₁₀ groups having one or more, e.g., 1,2, 3, 4, or 5, carbon-to-carbon double bonds. “Substituted alkene”refers to alkene substituted at 1 or more, e.g., 1, 2, 3, 4, or 5positions, which substituents are attached at any available atom toproduce a stable compound, with substitution as described herein.“Optionally substituted alkene” refers to alkene or substituted alkene.Likewise, “alkenylene” refers to divalent alkene. Examples of alkenyleneinclude without limitation, ethenylene (—CH═CH—) and all stereoisomericand conformational isomeric forms thereof. “Substituted alkenylene”refers to divalent substituted alkene. “Optionally substitutedalkenylene” refers to alkenylene or substituted alkenylene.

“Alkyne or “alkynyl” refers to a straight or branched chain unsaturatedhydrocarbon having the indicated number of carbon atoms and at least onetriple bond. Examples of a (C₂-C₈)alkynyl group include, but are notlimited to, acetylene, propyne, 1-butyne, 2-butyne, 1-pentyne,2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne, 2-heptyne,3-heptyne, 1-octyne, 2-octyne, 3-octyne and 4-octyne. An alkynyl groupcan be unsubstituted or optionally substituted with one or moresubstituents as described herein below. The term “alkynylene” refers todivalent alkyne. Examples of alkynylene include without limitation,ethynylene, propynylene. “Substituted alkynylene” refers to divalentsubstituted alkyne.

The term “alkoxy” refers to an —O-alkyl group having the indicatednumber of carbon atoms. For example, a (C₁-C₆)alkoxy group includes—O-methyl (methoxy), —O-ethyl (ethoxy), —O-propyl (propoxy),—O-isopropyl (isopropoxy), —O-butyl (butoxy), —O-sec-butyl (sec-butoxy),—O-tert-butyl (tert-butoxy), —O-pentyl (pentoxy), —O-isopentyl(isopentoxy), —O-neopentyl (neopentoxy), —O-hexyl (hexyloxy),—O-isohexyl (isohexyloxy), and —O-neohexyl (neohexyloxy). “Hydroxyalkyl”refers to a (C₁-C₁₀)alkyl group wherein one or more of the alkyl group'shydrogen atoms is replaced with an —OH group. Examples of hydroxyalkylgroups include, but are not limited to, —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH,—CH₂CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂CH₂CH₂OH, and branchedversions thereof. The term “ether” or “oxygen ether” refers to(C₁-C₁₀)alkyl group wherein one or more of the alkyl group's carbonatoms is replaced with an —O— group. The term ether includes—CH₂—(OCH₂—CH₂)_(q)OP₁ compounds where P₁ is a protecting group, —H, ora (C₁-C₁₀)alkyl. Exemplary ethers include polyethylene glycol,diethylether, methylhexyl ether and the like.

The term “thioether” refers to (C₁-C₁₀)alkyl group wherein one or moreof the alkyl group's carbon atoms is replaced with an —S— group. Theterm thioether includes —CH₂—(SCH₂—CH₂)_(q)—SP₁ compounds where P₁ is aprotecting group, —H, or a (C₁-C₁₀)alkyl. Exemplary thioethers includedimethylthioether, ethylmethyl thioether. Protecting groups are known inthe art and include, without limitation: 9-fluorenylmethyloxy carbonyl(Fmoc), t-butyloxycarbonyl (Boc), benzhydryloxycarbonyl (Bhoc),benzyloxycarbonyl (Cbz), O-nitroveratryloxycarbonyl (Nvoc), benzyl (Bn),allyloxycarbonyl (alloc), trityl (Trt), dimethoxytrityl (DMT),1-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl (Dde), diathiasuccinoyl(Dts), benzothiazole-2-sulfonyl (Bts) and monomethoxytrityl (MMT)groups.

“Aryl,” alone or in combination refers to an aromatic monocyclic orbicyclic ring system such as phenyl or naphthyl. “Aryl” also includesaromatic ring systems that are optionally fused with a cycloalkyl ring.A “substituted aryl” is an aryl that is independently substituted withone or more substituents attached at any available atom to produce astable compound, wherein the substituents are as described herein.“Optionally substituted aryl” refers to aryl or substituted aryl.“Arylene” denotes divalent aryl, and “substituted arylene” refers todivalent substituted aryl. “Optionally substituted arylene” refers toarylene or substituted arylene.

“Heteroatom” refers to N, O, P and S. Compounds that contain N or Satoms can be optionally oxidized to the corresponding N-oxide, sulfoxideor sulfone compounds. “Hetero-substituted” refers to an organic compoundin any embodiment described herein in which one or more carbon atoms aresubstituted with N, O, P or S.

“Cycloalkyl” refer to monocyclic, bicyclic, tricyclic, or polycyclic, 3-to 14-membered ring systems, which are either saturated, unsaturated oraromatic. The cycloalkyl group may be attached via any atom. Cycloalkylalso contemplates fused rings wherein the cycloalkyl is fused to an arylor hetroaryl ring. Representative examples of cycloalkyl include, butare not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.A cycloalkyl group can be unsubstituted or optionally substituted withone or more substituents as described herein below. “Cycloalkylene”refers to divalent cycloalkyl. The term “optionally substitutedcycloalkylene” refers to cycloalkylene that is substituted with 1, 2 or3 substituents, attached at any available atom to produce a stablecompound, wherein the substituents are as described herein.

“Carboxyl” or “carboxylic” refers to group having the indicated numberof carbon atoms and terminating in a —C(O)OH group, thus having thestructure —R—C(O)OH, where R is a divalent organic group that includeslinear, branched, or cyclic hydrocarbons. Non-limiting examples of theseinclude: C₁₋₈ carboxylic groups, such as ethanoic, propanoic,2-methylpropanoic, butanoic, 2,2-dimethylpropanoic, pentanoic, etc.

“(C₃-C₈)aryl-(C₁-C₆)alkylene” refers to a divalent alkylene wherein oneor more hydrogen atoms in the C₁-C₆ alkylene group is replaced by a(C₃-C₈)aryl group. Examples of (C₃-C₈)aryl-(C₁-C₆)alkylene groupsinclude without limitation 1-phenylbutylene, phenyl-2-butylene,1-phenyl-2-methylpropylene, phenylmethylene, phenylpropylene, andnaphthylethylene. The term “(C₃-C₈)cycloalkyl-(C₁-C₆)alkylene” refers toa divalent alkylene wherein one or more hydrogen atoms in the C₁-C₆alkylene group is replaced by a (C₃-C₈)cycloalkyl group. Examples of(C₃-C₈)cycloalkyl-(C₁-C₆)alkylene groups include without limitation1-cycloproylbutylene, cycloproyl-2-butylene,cyclopentyl-1-phenyl-2-methylpropylene, cyclobutyhnethylene andcyclohexylpropylene.

Unless otherwise indicated, the nucleic acids and nucleic acid analogsdescribed herein are not described with respect to any particularsequence of bases. The present disclosure is directed to divalentnucleobases, compositions comprising the divalent nucleobases, andmethods of use of the divalent nucleobases and compounds containingthose nucleobases, and the usefulness of any specific embodimentsdescribed herein, while depending upon a specific sequence in eachinstance, is generically applicable. Based on the abundance of publishedwork with nucleic acids, nucleic acid analogs and PNA (e.g., γPNA), itis expected that any nucleobase sequence attached to the backbone of thedescribed γPNA oligomers would hybridize in an expected, specific mannerwith a complementary nucleobase sequence of a target nucleic acid ornucleic acid analog by Watson-Crick or Watson-Crick-like hydrogenbonding. One of ordinary skill would understand that the compositionsand methods described herein are sequence-independent and describenovel, generalized compositions comprising divalent nucleobases andrelated methods.

In another embodiment, a genetic recognition reagent oligomer isprovided, comprising at least one divalent nucleobase. The geneticrecognition reagent comprises a backbone and at least two nucleobases,at least one of which is a divalent nucleobase as described herein. Thisstructure is shown schematically in Formula 3:

where R1 is a backbone monomer residue and R2s are, independentlynucleobases, where at least one instance of R2 is a divalent nucleobase,such as any one of JB1-JB16 as shown herein. E are independently end(terminal) groups that are part of the terminal monomer residues, and“n” is any positive integer or 0, for example 48 or less, 28 or less, 23or less, and 18 or less, including 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, and 18. Typically, all instances of R1 are thesame with the exception of the terminal monomer residues which typicallyhave different end-groups E as compared to internal monomers, such as,without limitation NH₂ and C(O)OH or CONH₂ at the respective N-terminaland C-terminal ends for PNAs, and hydroxyl groups at the 5′ and 3′ endsof nucleic acids.

In one embodiment of the present invention, the genetic recognitionreagents are implemented on an array. Arrays are particularly useful inimplementing high-throughput assays, such as genetic detection assays.As used herein, the term “array” refers to reagents, for example thegenetic recognition reagents described herein, located at two or morediscrete, identifiable and/or addressable locations on a substrate. Inone embodiment, an array is an apparatus having two or more discrete,identifiable reaction chambers, such as, without limitation a 96-welldish, in which reactions comprising identified constituents areperformed. In an exemplary embodiment, two or more genetic recognitionreagents described herein are immobilized onto a substrate in aspatially addressable manner so that each individual primer or probe islocated at a different and (addressable) identifiable location on thesubstrate. Substrates include, without limitation, multi-well plates,silicon chips and beads. In one embodiment, the array comprises two ormore sets of beads, with each bead set having an identifiable marker,such as a quantum dot or fluorescent tag, so that the beads areindividually identifiable using, for example and without limitation, aflow cytometer. In one embodiment, an array is a multi-well platecontaining two or more wells with the descried genetic recognitionreagents for binding specific sequences. As such, reagents, such asprobes and primers may be bound or otherwise deposited onto or intospecific locations on an array. Reagents may be in any suitable form,including, without limitation: in solution, dried, lyophilized orglassified. When linked covalently to a substrate, such as an agarosebead or silicon chip, a variety of linking technologies are known forattaching chemical moieties, such as the genetic recognition reagents tosuch substrates. Linkers and spacers for use in linking nucleic acids,peptide nucleic acids and other nucleic acid analogs are broadly knownin the chemical and array arts and for that reason are not describedherein. As a non-limiting example, a γPNA genetic recognition reagentcontains a reactive amine, which can be reacted with carboxyl, cyanogenbromide-, N-hydroxysuccinimide ester-, carbonyldiimidazole- oraldehyde-functional agarose beads, available, for instance from ThermoFisher Scientific (Pierce Protein Biology Products), Rockford, Ill. anda variety of other sources. The genetic recognition reagents describedherein can be attached to a substrate in any manner, with or withoutlinkers. Informatics and/or statistical software or othercomputer-implemented processes for analyzing array data and/oridentifying genetic risk factors from data obtained from a patientsample, are known in the art.

Certain of the JB divalent compositions are expected to exhibitfluorescence, such as JB1, JB2 and JB3, due to their ring structure.These compositions can, of course, be used as fluorochromes, or theintrinsic fluorescence can be employed as a probe, for example, bybinding target sequences in an in situ assay or in a gel or blot, suchthat a target sequence can be visualized.

Thus, according to one embodiment of the present invention, a method isprovided for detection of a target sequence in a nucleic acid,comprising contacting a genetic recognition reagent composition asdescribed herein with a sample comprising nucleic acid and detectingbinding of the genetic recognition reagent with a nucleic acid. In oneembodiment, the genetic recognition reagent is immobilized on asubstrate, for example in an array, and labeled (e.g., fluorescentlylabeled or radiolabeled) nucleic acid sample is contacted with theimmobilized genetic recognition reagent and the amount of labelednucleic acid specifically bound to the genetic recognition reagent ismeasured. In a variation, genetic recognition reagent or a nucleic acidcomprising a target sequence of the genetic recognition reagent is boundto a substrate, and a labeled nucleic acid comprising a target sequenceof the genetic recognition reagent or a labeled genetic recognitionreagent is bound to the immobilized genetic recognition reagent ornucleic acid, respectively to form a complex. In one embodiment, thenucleic acid of the complex comprises a partial target sequence so thata nucleic acid comprising the full target sequence would out-compete thecomplexed nucleic acid for the genetic recognition reagent. The complexis then exposed to a nucleic acid sample and loss of bound label fromthe complex could be detected and quantified according to standardmethods, facilitating quantification of a nucleic acid marker in thenucleic acid sample. These are merely two of a large number of possibleanalytical assays that can be used to detect or quantify the presence ofa specific nucleic acid in a nucleic acid sample.

By “immobilized” in reference to a composition such as a nucleic acid orgenetic recognition reagent as described herein, it is meant attached toa substrate of any physical structure or chemical composition. Theimmobilized composition is immobilized by any method useful in thecontext of the end use. The composition is immobilized by covalent ornon-covalent methods, such as by covalent linkage of amine groups to alinker or spacer, or by non-covalent bonding, including Van derWaalsand/or hydrogen bonding. A “label” is a chemical moiety that is usefulin detection of, or purification or a molecule or composition comprisingthe label. A label may be, for example and without limitation, aradioactive moiety, such as ¹⁴C, ³²P, ³⁵S, a fluorescent dye, such asfluorescein isothiocyanate or a cyanine dye, an enzyme, or a ligand forbinding other compounds such as biotin for binding streptavidin, or anepitope for binding an antibody. A multitude of such labels, and methodsof use thereof are known to those of ordinary skill in the immunologyand molecular biology arts.

In yet another embodiment of the present invention, a method ofisolation and purification or a nucleic acid containing a targetsequence is provided. In one non-limiting embodiment, a geneticrecognition reagent as described herein is immobilized on a substrate,such as a bead (for example and without limitation, an agarose bead, abead containing a fluorescent marker for sorting, or a magnetic bead),porous matrix, surface, tube, etc. A nucleic acid sample is contactedwith the immobilized genetic recognition reagent and nucleic acidscontaining the target sequence bind to the genetic recognition reagent.The bound nucleic acid is then washed to remove unbound nucleic acids,and the bound nucleic acid is then eluted, and can be precipitated orotherwise concentrated by any useful method as are broadly known in themolecular biological arts.

In a further embodiment, kits are provided. A kit comprises at a minimuma vessel of any form, including cartridges for automated PNA synthesis,which may comprise one or more vessels in the form of compartments.Vessels may be single-use, or contain sufficient contents for multipleuses. A kit also may comprise an array. A kit may optionally compriseone or more additional reagents for use in making or using geneticrecognition reagents in any embodiment described herein. The kitcomprises a vessel containing any divalent nucleobase described herein,or monomers or genetic recognition reagents according to any embodimentdescribed herein. Different nucleobases, monomers or genetic recognitionreagents are typically packages into separate vessels, which may beseparate compartments in a cartridge.

EXAMPLES

The examples described herein illustrate advantages of the disclosedtechnology over traditional oligonucleotide systems. γPNA containingJB1-4 as recognition elements (γPNAJB1-4) can be designed to bind to anysequence of double-stranded DNA (dsDNA) or RNA (dsRNA) underphysiological conditions through direct hydrogen bonding interactions.These probes typically are as short 3 nt in length and as long as 25 ntin length. The shorter probes, 10 nt or less, are especially easier tosynthesize and scale up, via solution—as opposed to solid-phasechemistry, and are more readily taken up by cells.

Recognition of dsDNA or dsRNA by γPNAJB1-4 is more sequence-specificthan the existing antigene or antisense platforms because a mismatch onone face of JB would result in a mismatch on the second face. Binding ofγPNAJB1-4 to DNA or RNA is more selective for double-strand than forsingle-strand or primary (unstructured) sequences. Additionally, γPNAcontaining JB 1-16 (γPNAJB1-16) can be designed to bind to any secondaryor tertiary structure of RNA and modulate its folding patterns andthree-dimensional architectures.

Furthermore, these examples also provide distinctive features andadvantages over small-molecule drugs or other ligands. Recognition ofthe γPNAJB probes occurs in a sequence-specific and predictable mannerin accordance with the Watson-Crick and Janus base-pairing rules.Because they are modular in nature, γPNAJB probes could be modified tomatch the sequence of any RNA. This is an essential requirement forcountering drug resistance due to emergence of genetic mutations—inparticular, for genetic targets, such as that of cancer, bacteria, viraland parasite genomes or transcriptomes.

Example applications include, but are not limited to, reagents fordetection, analysis and purification of DNA and RNA molecules, and formolecular tools for regulating gene expression, correcting geneticmutations, modulating RNA splicing, controlling microRNA functions, andmodulation of RNA folding patterns and architectures. These applicationswill positively contribute to diagnostics; for example to detect DNA andRNA sequence and structural variations. Additionally this platform willadvance medicine; as an example for use as therapeutics for treating avariety of genetic diseases associated with misregulation of geneexpression including cancer, deregulation of RNA splicing, DNA and RNAunstable repeats, and fungal, bacterial and viral biology andinfections.

The applications of this nucleic acid platform using γPNA and the Janusnucleobases span basic research in biology and biotechnology,diagnostics, and therapeutics. This platform, which targetsdouble-stranded DNA and RNA has been demonstrated and described inaccordance with several examples, which are intended to be illustrativein all aspects rather than restrictive. Thus, the present invention iscapable of many variations in detailed implementation, which may bederived from the description contained herein by a person of ordinaryskill in the art.

Example 1 Computer Simulation of Interactions of γPNA with DNA and RNA

DNA binding properties of γPNA include efficiencies from backbonepreorganization (as defined by the stereochemistry at the γ-position,which determines the helical sense of the oligomer) and improvements inhydrogen-bonding and base-stacking capabilities. Divalent nucleobases JB1-4, were designed to bind to the perfectly matched, and JB 5-16 weredesigned to bind to mismatched base-pairs in the DNA or RNA doublehelix. In both cases binding occurs via strand invasion, whereby oneface of the divalent nucleobase forms Watson-Crick-like hydrogen-bondinginteractions with natural bases on one strand of DNA or RNA doublehelix, while a second face forms ‘Janus’ hydrogen-bonding interactionswith the complementary strand of the DNA or RNA target. As indicatedabove, FIG. 1 illustrates these hydrogen-bonding interactions, whilecomparing to Watson-Crick-like hydrogen-bonding interactions withnatural bases.

In one embodiment, the overall geometry, bond distance, and chemicalfunctionalities of these divalent “Janus” nucleobases are carefullycrafted so that they can be used interchangeably to target theperfectly-matched as well as mismatched binding sites, or a combinationthereof. It is demonstrated from these examples that this platform ofγPNA monomers/oligomers containing the JBs are more selective fordouble-stranded DNA or RNA, in addition to recognizing secondary andtertiary structures. DNA-γPNA interactions were modeled by computersimulation using AMBER10 (University of California, San Francisco,Calif.). The γPNA is based on alanine, having the monomer structure

and thus having the residue structure

FIG. 5 illustrates the simulated structures of γPNAJB1-4 invading DNAdouble helix.

FIG. 6 illustrates the structures of γPNAJB1-16 invading an RNAsecondary structure containing perfectly-matched and mismatchedbase-pairs.

Example 2 Synthesis of Divalent Nucleobases, Nucleotide Monomers andγPNA Oligomers

Examples of reactions performed to synthesis JBs 1-16 are shown in FIG.7. Upon synthesis of these specialized bases, the corresponding monomerscan be synthesized and an example synthesis scheme is described in FIG.8. FIG. 9 illustrates the synthesis of oligomers containing the γPNAoligomers.

The present invention has been described with reference to certainexemplary embodiments, dispersible compositions and uses thereof.However, it will be recognized by those of ordinary skill in the artthat various substitutions, modifications or combinations of any of theexemplary embodiments may be made without departing from the spirit andscope of the invention. Thus, the invention is not limited by thedescription of the exemplary embodiments, but rather by the appendedclaims as originally filed.

We claim:
 1. A genetic recognition reagent comprising a plurality ofnucleobase residues attached to a nucleic acid or nucleic acid analogbackbone, in which at least one nucleobase residue is a divalentnucleobase chosen from the following:

in which each instance of R₁ is, independently, a protecting group or Hand X is CH or N.
 2. The genetic recognition reagent of claim 1, inwhich each instance of R₁ is, independently, H or a protecting groupchosen from: methyl, formyl, ethyl, acetyl, anisyl, benzyl, benzoyl,carbamate, trifluoroacetyl, diphenylmethyl, triphenylmethyl,N-hydroxysuccinimide, benzyloxymethyl, benzyloxycarbonyl,2-nitrobenzoyl, t-Boc (tert-butyloxycarbonyl), 4-methylbenzyl,4-nitrophenyl, 2-chlorobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl,2,4,5-trichlorophenyl, thioanizyl, thiocresyl, cbz (carbobenzyloxy),p-methoxybenzyl carbonyl, 9-fluorenylmethyloxycarbonyl,pentafluorophenyl, p-methoxybenzyl, 3,4-dimethozybenzyl,p-methoxyphenyl, 4-toluenesulfonyl, p-nitrobenzenesulfonates,9-fluorenylmethyloxycarbonyl, 2-nitrophenylsulfenyl,2,2,5,7,8-pentamethyl-chroman-6-sulfonyl, and p-bromobenzenesulfonyl. 3.The genetic recognition reagent of claim 1, in which the backbone ischosen from one of a DNA, RNA, peptide nucleic acid (PNA),phosphorothioate, locked nucleic acid, unlocked nucleic acid,2′-O-methyl-substituted RNA, morpholino nucleic acid, threose nucleicacid, or glycol nucleic acid backbone, or any combination thereof. 4.The genetic recognition reagent of claim 1, in which the backbone is apeptide nucleic acid (PNA) backbone.
 5. The genetic recognition reagentof claim 1, in which the backbone is a gamma peptide nucleic acid (γPNA)backbone.
 6. The genetic recognition reagent of claim 5, in which thebackbone is PEGylated, with one or more PEG moieties of two to fifty(—O—CH₂—CH₂—) residues.
 7. The genetic recognition reagent of claim 1,in which the backbone is a γPNA backbone in which a backbone monomerresidue of the γPNA backbone is

wherein R is a nucleobase residue, R₁ of the backbone monomer residue,R₂and R₃ are, independently, H, amino acid side chains, linear orbranched (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₈)hydroxyalkyl, (C₃-C₈)aryl, (C₃-C₈)cycloalkyl,(C₃-C₈)aryl(C₁-C₆)alkylene, (C₃-C₈)cycloalkyl(C₁-C₆)alkylene, PEGylatedmoieties of the preceding comprising from 1 to 50 (—O—CH₂—CH₂—)residues, —CH₂—(OCH₂—CH₂)_(q)OP₁, —CH₂—(OCH₂—CH₂)_(q)—NHP₁,—CH₂—(OCH₂—CH₂)_(q)—SP₁ and —CH₂—(SCH₂—CH₂)_(q)—SP₁,—CH₂—(OCH₂—CH₂)_(r)—OH, —CH₂—(OCH₂—CH₂)_(r)—NH₂,—CH₂—(OCH₂—CH₂)_(r)—NHC(NH)NH₂, or—CH₂—(OCH₂—CH₂)_(r)—S—S[CH₂CH₂]_(s)NHC(NH)NH₂, where P₁ is selected fromthe group consisting of H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₃-C₈)aryl, (C₃-C₈)cycloalkyl, (C₃-C₈)aryl(C₁-C₆)alkylene and(C₃-C₈)cycloalkyl(C₁-C₆)alkylene; q is an integer from 0 to 10,inclusive; r and s are each independently integers from 1 to 50,inclusive; where R₁ of the backbone monomer residue and R₂ are differentand one of R₁ of the backbone monomer residue or R₂is H.
 8. The geneticrecognition reagent of claim 7, in which R₂ is H, R₁ of the backbonemonomer residue is an amino acid side chain that is optionallyPEGylated, with one or more PEG moieties of one to twelve (—O—CH₂—CH₂—)residues.
 9. The genetic recognition reagent of claim 1, in which thenucleobase residues are arranged in a sequence complementary to a targetsequence of a nucleic acid.
 10. The genetic recognition reagent of claim1, having from 3 to 25 nucleobase residues.
 11. The genetic recognitionreagent of claim 1, having the structure:

where each instance of R₄ is a backbone monomer residue and eachinstance of R is one of the plurality of nucleobase residues, where atleast one instance of R the divalent nucleobase, E are independently endgroups, and “n” is zero or a positive integer ranging from 1 to 48,wherein the plurality of nucleobase residues forms a sequence that iscomplementary to a target sequence of a nucleic acid.
 12. The geneticrecognition reagent of claim 1, in which the divalent nucleobase ischosen from JB1, JB2, JB3, or JB4.
 13. The genetic recognition reagentof claim 1, in which all instances of R₁ are H.
 14. A monomer forproduction of a genetic recognition reagent comprising a backbonemonomer for a genetic recognition reagent covalently attached to adivalent nucleobase chosen from the following:

in which each instance of R₁ is, independently, a protecting group or Hand X is CH or N.
 15. The monomer of claim 14, in which the nucleobaseis one of JB1, JB2, JB3 or JB4.
 16. The monomer of claim 14, in which R₁is a protecting group.
 17. The monomer of claim 16, in which theprotecting group is chosen from one or more of: methyl, formyl, ethyl,acetyl, anisyl, benzyl, benzoyl, carbamate, trifluoroacetyl,diphenylmethyl, triphenylmethyl, N-hydroxysuccinimide, benzyloxymethyl,benzyloxycarbonyl, 2-nitrobenzoyl, t-Boc (tert-butyloxycarbonyl),4-methylbenzyl, 4-nitrophenyl, 2-chlorobenzyloxycarbonyl,2-bromobenzyloxycarbonyl, 2,4,5-trichlorophenyl, thioanizyl, thiocresyl,cbz (carbobenzyloxy), p-methoxybenzyl carbonyl,9-fluorenylmethyloxycarbonyl, pentafluorophenyl, p-methoxybenzyl,3,4-dimethozybenzyl, p-methoxyphenyl, 4-toluenesulfonyl,p-nitrobenzenesulfonates, 9-fluorenylmethyloxycarbonyl,2-nitrophenylsulfenyl, 2,2,5,7,8-pentamethyl-chroman-6- sulfonyl, andp-bromobenzenesulfonyl.
 18. The monomer of claim 14, in which themonomer is a peptide nucleic acid (PNA) monomer.
 19. The monomer ofclaim 14, in which the monomer is a gamma peptide nucleic acid (γPNA)backbone monomer.
 20. The monomer of claim 14, in which the backbonemonomer is PEGylated, with one or more PEG moieties of two to fifty(—O—CH₂—CH₂—) residues.
 21. The monomer of claim 14, in which themonomer is a γPNA monomer having the structure:

wherein R is a nucleobase residue, R₁ of the γPNA monomer, R₂ and R₄are, independently, H, amino acid side chains, linear or branched(C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)aryl, (C₃-C₈)cycloalkyl, (C₃-C₈)aryl(C₁-C₆)alkylene,(C₃-C₈)cycloalkyl(C₁-C₆)alkylene, PEGylated moieties of the precedingcomprising from 1 to 50 (—O—CH₂—CH₂—) residues, —CH₂—(OCH₂—CH₂)_(q)OP₁,—CH₂—(OCH₂—CH₂)_(q)—NHP₁, —CH₂—(OCH₂—CH₂)_(q)—SP₁ and—CH₂—(SCH₂—CH₂)_(q)—SP₁, —CH₂—(OCH₂—CH₂)_(r)—OH,—CH₂—(OCH₂—CH₂)_(r)—NH₂, —CH₂—(OCH₂—CH₂)_(r)—NHC(NH)NH₂, or—CH₂—(OCH₂—CH₂)_(r)—S—S[CH₂CH₂]_(s)NHC(NH)NH₂, where P₁ is selected fromthe group consisting of H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₃-C₈)aryl, (C₃-C₈)cycloalkyl, (C₃-C₈)aryl(C₁-C₆)alkylene and(C₃-C₈)cycloalkyl(C₁-C₆)alkylene; q is an integer from 0 to 10,inclusive; r and s are each independently integers from 1 to 50,inclusive; where R₁ of the γPNA monomer and R₂ are different, and one ofR₁ of the γPNA monomer or R₂ is H, and R₃ is H or a protecting group.22. The monomer of claim 21, in which R₂ is H, R₁ of the γPNA monomer isan amino acid side chain that is optionally PEGylated, with one or morePEG moieties of one to twelve (—O—CH₂—CH₂—) residues, and R₃ is aprotecting group.
 23. A nucleobase chosen from one the following:

in which each instance of R₁ is, independently a protecting group or H,R is a reactive group and X is CH or N.
 24. The nucleobase of claim 23,chosen from one of the following:

in which each instance of R₁ is, independently a protecting group or H.25. A kit comprising a monomer of claim 14 in a vessel.
 26. The kit ofclaim 25, further comprising monomers comprising each of JB1, JB2, JB3,JB4,

where R₁ is a protecting group, or H,

JB7, JB8, JB9, JB9b, JB10, JB11, JB12, JB13, JB14, JB15, and JB16nucleobases in separate vessels.
 27. An array comprising the geneticrecognition reagent of claim
 1. 28. A method of detection of a targetsequence in a nucleic acid, comprising contacting the geneticrecognition reagent of claim 1 with a sample comprising nucleic acid anddetecting binding of the genetic recognition reagent with the nucleicacid.
 29. A method of isolation and purification of a nucleic acidcontaining a target sequence, comprising, contacting a nucleic acidsample with the genetic recognition reagent of claim 1, separating thenucleic acid sample from the genetic recognition reagent, leaving anynucleic acid bound to the genetic recognition reagent bound to thegenetic recognition reagent, and separating the genetic recognitionreagent from any nucleic acid bound to the genetic recognition reagent.30. The method of claim 29 in which the genetic recognition reagent isimmobilized on a substrate, comprising contacting the nucleic acid withthe substrate, washing the substrate to remove unbound nucleic acid fromthe substrate, but leaving bound nucleic acid bound to the substrate,and eluting the bound nucleic acid from the substrate.
 31. The geneticrecognition reagent of claim 1, wherein at least one nucleobase is JB1.32. The genetic recognition reagent of claim 1, wherein at least onenucleobase is JB2.
 33. The genetic recognition reagent of claim 1,wherein at least one nucleobase is JB3.
 34. The genetic recognitionreagent of claim 1, wherein at least one nucleobase is JB4.
 35. Agenetic recognition reagent comprising a plurality of nucleobaseresidues attached to a peptide nucleic acid backbone, in which at leastone nucleobase is

where R₁ is a protecting group, or H.
 36. The genetic recognitionreagent of claim 35, wherein the backbone is a gamma peptide nucleicacid (γPNA) backbone.
 37. The monomer of claim 14, wherein the divalentnucleobase is JB1.
 38. The monomer of claim 14, wherein the divalentnucleobase is JB2.
 39. The monomer of claim 14, wherein the divalentnucleobase is JB3.
 40. The monomer of claim 14, wherein the divalentnucleobase is JB4.
 41. The nucleobase of claim 23, having the structureof JB1.
 42. The nucleobase of claim 23, having the structure of JB2. 43.The nucleobase of claim 23, having the structure of JB3.
 44. Thenucleobase of claim 23, having the structure of JB4.
 45. The nucleobaseof claim 23, wherein R is carboxyl.
 46. The kit of claim 25, wherein themonomer comprises a peptide nucleic acid backbone monomer.
 47. The kitof claim 25, wherein the monomer comprises a γ-peptide nucleic acidbackbone monomer.